Download Transport to the vacuole: receptors and trans elements

Journal of Experimental Botany, Vol. 49, No. 325, pp. 1271–1279, August 1998
Transport to the vacuole: receptors and trans elements
Leonard Beevers1 and Natasha V. Raikhel2,3
1 Department of Botany and Microbiology, University of Oklahoma, Norman, Oklahoma, USA
2 MSU DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA
Received 2 March 1998; Accepted 30 March 1998
Abstract
Most proteins that are synthesized on membranebound ribosomes are transported through the Golgi
and reach the trans-Golgi network to be sorted for
delivery to various cellular destinations, including the
vacuole. Sorting involves a recognition of proteins by
receptors and the assembly of cytosol-oriented coat
structures that package cargo into vesicles. Vesicle
trafficking is regulated by specific membrane-bound
and soluble proteins. Several components of the
secretory machinery have recently been identified in
plants and are described in this review. Ongoing and
future research will characterize features of the
secretory pathway specific to plants which, because
of the multiplicity of vacuole types, provide a more
complex paradigm than the better described mammalian and yeast systems.
Key words: Clathrin coated vesicles, protein trafficking,
receptors, secretory pathway, SNAREs, vesicles.
Introduction
Previous articles in this series have established the role of
early components of the endomembrane system, endoplasmic reticulum and Golgi apparatus, in the synthesis
and processing of proteins destined for secretion
(Andreeva et al., 1998; Okita, 1998; Vitale and Denecke,
1998). After arrival in the Golgi apparatus, proteins are
selectively retrieved for eventual transfer to such other
cellular compartments as protein storage vacuoles, lytic
vacuoles, vacuolar membranes, the plasma membrane or
the cell exterior. The current understanding of the basis
for selectivity to the various destinations is, in most cases,
somewhat limited. It appears that delivery of soluble
proteins to the cell exterior is by a default mechanism
(Chrispeels, 1991) whereas transfer to the internal lytic
3 To whom correspondence should be addressed. Fax: +1 517 353 9168.
© Oxford University Press 1998
and storage vacuoles is dependent upon specific targeting
information. In mammalian systems, the targeting of
soluble proteins to the lytic compartment, the lysosome,
is frequently mediated by mannose 6-phosphate residues
in glycosyl side-chains of the glycoproteins ( Kornfeld,
1990). Vacuolar acid hydrolases in plant systems are also
glycoproteins (Gaudreault and Beevers, 1983); however,
plants do not contain mannose 6-phosphate residues or
M-6-P receptors (Gaudreault and Beevers, 1984). Instead
the targeting of soluble proteins and some storage proteins
to the vacuole is mediated by short peptide sequences in
the protein or precursor proteins. These targeting signals
can occur at the amino terminus of propeptides (NTPP)
or at the carboxy terminus (CTPP), or they may be part
of the mature protein (Chrispeels and Raikhel, 1992).
The role of these peptide sequences is described in detail
in the mini review by Nakamura and Neuhaus (1998).
In mammalian systems, the functioning of M-6-P as a
targeting determinant is dependent upon the ability of
the ligand to interact with receptors that subsequently
permit the sequestration of the proteins containing
mannose 6-phosphate residues. Interaction of ligand
and receptor enables sorting of such proteins from the
Golgi apparatus and eventual delivery to the lysosome
( Kornfeld and Mellman, 1989). This system has provided
the paradigm for lysosomal sorting, and by analogy it
might be anticipated that the targeting determinants in
plant vacuolar proteins would similarly react with receptors, thus permitting selection within the Golgi apparatus
of proteins eventually delivered to the vacuole. In this
review, the search for and identification of such receptors
is described and their involvement in delivery of proteins
to the vacuole is characterized.
Identification of receptors from plants
In mammalian systems the M-6-P receptor involved in
the recovery of lysosomal precursors from the trans-Golgi
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becomes incorporated into clathrin coated vesicles (CCVs)
(Leborgne and Hoflack, 1997). By analogy, it was reasoned that a receptor for the targeting determinants for
vacuolar delivery in plants would also be present in
CCVs. This contention was consistent with an earlier
observation that CCVs from developing pea cotyledon
contained acid hydrolases (Harley and Beevers, 1989)
normally associated with the vacuole. When detergent
extracts from CCVs were applied to affinity columns
prepared with the targeting determinant of propeptides
of barley aleurain an 80 kDa protein was retained ( Kirsch
et al., 1994). The retained protein was eluted at pH 4.0.
Affinity columns prepared with sequences not involved in
targeting did not retain the 80 kDa protein from the
CHAPS extract. The 80 kDa protein was not retained on
affinity columns with the C terminal targeting determinant
of barley lectin; this observation is consistent with the
hypothesis that there may be several pathways by which
proteins are selected and delivered to the vacuole
(Matsuoka et al., 1995). Binding of the 80 kDa protein
to the proaleurain targeting peptide was inhibited by the
NTPP of prosporamin. Similarly, the 80 kDa protein in
detergent extracts of CCVs was retained on affinity columns containing the prosporamin targeting sequence
( Kirsch et al., 1996). Binding of the 80 kDa protein to
the prosporamin and proaleurain targeting sequences
involves the NPIR motif common to both protein precursors. Mutants of the prosporamin sequence in which
the I is replaced by G in the NPIR motif failed to bind
the 80 kDa protein and were non-functional in targeting
sporamin to vacuoles in transgenic constructs ( Kirsch
et al., 1996). With regard to functioning in targeting, the
location of the NPIR motif in the protein appears to be
of little consequence. Koide et al. (1997) have demonstrated that attachment of NPIR motifs at the carboxy
terminus in transgenic constructs results in targeting of
protein to the vacuole. A survey of many vacuolar
proteins demonstrated the occurrence of internal NPIR
sequences which, in all likelihood, are the functional
targeting determinants (Nakamura and Neuhaus, 1997).
In this regard the binding of targeting sequences to the
identified 80 kDa receptor from CCVs does not appear
to depend on location. The storage protein 2S albumin
of Brazil nut is targeted to the vacuole by sequences
located partially in an excised carboxy presequence and
the mature protein. The carboxy targeting sequence does
not contain an NPIR motif; however the 80 kDa protein
from CHAPS extracts of CCVs binds to the C-terminal
peptide. Modifications of the peptide sequences that result
in mistargeting of proteins (secretion) result in failure to
bind the 80 kDa protein ( Kirsch et al., 1996). Collectively,
these results demonstrate that the capacity of targeting
sequences to bind to the 80 kDa protein is closely linked
to their ability to function in vacuolar targeting and
supports the role of the 80 kDa protein as a receptor for
sorting vacuolar proteins.
Protease treatment of intact CCVs followed by CHAPS
extraction and affinity purification demonstrated that
approximately 5 kDa of the protein’s C-terminus was
accessible on the cytoplasmic surface of the CCVs,
whereas the N-terminal intraluminal protein carried the
ligand-binding domain ( Kirsch et al., 1994). Subsequent
molecular cloning has identified four homologues of the
80 kDa protein from cDNA libraries from peas. Two
homologues have been identified in Arabidopsis and one
in maize (Paris et al., 1997). In the most extensively
characterized system from peas cDNA NP471, Gen Bank
U79958 codes for a protein of 623 amino acids (Paris
et al., 1997). The first 22 amino acids represent a signal
peptide and there is a single 21-residue hydrophobic
region. The cDNA sequence predicts a Type I transmembrane protein with a large N-terminal luminal domain
containing three potential N glycosylation sites and an
estimated 4.6 kDa C-terminal domain of 38 amino acids.
Within the N-terminal domain the first 400 amino acids
represent a unique region with no homology to yeast or
animal sequences in the current gene databases. This
region does show homology to sequences from maize,
rice, and Arabidopsis and within pea and Arabidopsis
there appears to be gene families for the protein. The
remaining intraluminal region of the 80 kDa protein is
occupied by the Epidermal Growth Factor ( EGF ) cysteine-rich repeats. The first two repeats demonstrate the
B1 motif and the third has a B2 motif characteristic of
the EGF repeats identified in several receptors from
mammalian sources (Herz et al., 1988; Campbell and
Bork, 1993). The three cysteine-rich EGF repeats are
positioned between the unique region and a short SerThr region that precedes the transmembrane domain. The
Ser-Thr is identified as a potential O-glycosylation site in
the Arabidopsis homologue (Ahmed et al., 1997). The
EGF repeats have been implicated in calcium-dependent
protein–protein interactions in receptors from animal
systems. It is significant that the affinity chromatography
that identified the 80 kDa binding protein was conducted
in the presence of 1 mM CaCl and that within the third
2
EGF repeat of the 80 kDa protein are conserved residues
considered to be important in calcium binding (Rao et al.,
1995; Downing et al., 1996). Partial digestion of the
80 kDa protein with clostripain releases a series of fragments only a few of which are retained on the affinity
column containing the NPIR sequence. The fragments
binding to the affinity matrix originate from a site adjacent
to the transmembrane domain rather than the unique
region. This observation suggests that, similar to mammalian systems, sequences close to the EGF region of the
plant 80 kDa protein may be involved in ligand binding
( T Kirsch and L Beevers, unpublished results).
By screening the EST database for EGF receptor repeat
The trans-Golgi to vacuole 1273
Fig. 1. Predicted structure of the AtELP gene product. The predicted
N-terminal signal sequence (SS), the EGF repeats, the transmembrane
domain, and cytoplasmic tyrosine motif are depicted. Reprinted from
Ahmed et al. (1997) with permission from the American Society of
Plant Physiologists.
sequence, Ahmed et al. (1997) have identified a protein
that shows common features with the sorting receptors
of mammalian and yeast cells. Although no function was
characterized for the protein, it is a transmembrane Type I
protein with three EGF repeats (Fig. 1) and shows 72%
identity to the 80 kDa protein NP471 from pea cotyledons. It was associated with two membrane fractions,
one enriched in clathrin and its associated adaptor
containing vesicles and an unidentified compartment.
An isoform of the pea 80 kDa receptor-like protein has
been identified in the Cambridge two-dimensional PAGE
database of Arabidopsis membrane proteins (Dupree,
personal communication).
More recently, Shimada et al. (1997) have isolated two
potential vacuolar sorting receptors (of 72 kDa and
82 kDa) for vacuolar proteins from dense vesicles of
developing pumpkin cotyledons. Similar to the 80 kDa
receptor from peas, the receptor from pumpkin binds to
the NPIR sequence of proaleurain and sweet potato
prosporamin and an internal peptide of NPWR. A cDNA
for the 72 kDa protein, isolated from a pumpkin seed
cDNA library, encodes a protein of 624 amino acids that
conforms to a Type I integral membrane protein. The
protein has a 549-residue luminal domain containing
three EGF-like motifs in the C-terminal region proximal
to a 17 amino acid transmembrane domain and a cytoplasmic tail of 37 amino acids.
The isolation of the 80 kDa binding protein in CCVs
implicates these organelles in the transport of vacuolar
proteins. The characterization of the protein as a Type I
membrane protein is consistent with the selective function
of the binding protein in the trans-Golgi with subsequent
sequestration of the binding protein and bound cargo
protein into CCVs. Ultrastructural studies demonstrating
the occurrence of the binding protein in distended Golgi
cisternae are consistent with this concept (Paris et al.,
1997).
Vesicle assembly-adaptors
The mechanism of retrieval of the binding protein and
ligated cargo protein from the trans-Golgi is not resolved.
However, association of the 80 kDa binding protein from
a less dense membrane fraction with components released
from CCVs by TRIS washing has been demonstrated
( Kirsch et al., 1994). The TRIS washes contain the
clathrin coat components comprised of clathrin heavy
and light chains and adaptors (Beevers, 1996; Robinson,
1996). Adaptors characterized from mammalian systems
are complex heterotetramers that couple the assembly of
clathrin vesicles with the entrapment of membrane receptors (Schmidt, 1997; Kirchhausen et al., 1997). Endocytic
coated pits and CCVs formed at the plasma membrane
contain the AP-2 adaptor complex whereas buds and
coated vesicles derived from the trans-Golgi contain the
related AP-1 complex. The AP-2 complex contains two
large#100 kDa chain adaptors (one a chain and one b 1
or b 2 chain), a medium 50 kDa m2 chain and a small
17 kDa s2 chain. AP-1 contains c and b1 adaptors of
#100 kDa, together with a medium m1 (47 kDa) and a
small 19 kDa s1 chain (Pearse and Robinson, 1990; Keen,
1990). A preliminary characterization of adaptors from
developing pea cotyledons suggests a preponderance of
AP-1 and the presence of only one b component, which
cross-reacts with monoclonal antibodies to the mammalian b adaptor (Holstein et al., 1994). Other adaptor
components from plants have shown no cross-reaction to
antibodies to other mammalian adaptor subunits (Butler
and Beevers, 1994), although molecular studies have
demonstrated that the deduced amino acid sequence of
cDNA of the 19 kDa s1 from plants shows over 70%
similarity to mammalian homologues. The studies indicate
that there are multicopies of the AP-19 gene present,
raising the possibility of cell and tissue specific expression
of genes with isoforms of the protein product in individual
cells or tissues (Moldonado-Mendoza and Nessler, 1996;
Moldonado-Mendoza et al., 1996).
The current view for the recruitment of clathrin coats
to vesicles in mammalian systems is that APs are first
recruited from the cytosol to the membrane ( Kirchhausen
et al., 1997). This recruitment involves the interaction of
APs with receptor tails. The cytosolic tails of receptors
from mammalian systems have been shown to carry
specific signals that direct their internalization and are
involved in other stages of intracellular targeting
(Sandoval and Bakke, 1994). Specifically, the best characterized sequences have either a critical tyrosine residue or
a pair of leucine or bulky hydrophobic residues. It has
been demonstrated that the tyrosine-based signals may
bind directly to adaptor complexes, particularly the AP2 (Sosa et al., 1993; Glickman et al., 1989; Ohno et al.,
1995; Kirchhausen et al., 1997). In some instances both
AP-1 and AP-2 have been demonstrated to bind to
tyrosine-based motifs; in other instances tyrosine does
not appear to be involved in AP-1 binding (Honing
et al., 1997).
A survey of the C-terminal cytosolic domain of the
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Beevers and Raikhel
80 kDa protein, the receptor-like protein from
Arabidopsis, and pumpkin indicates the presence of tyrosine-based signal sequences that could be involved in
recruiting the adaptor proteins into vesicles. In this context it has been demonstrated that in a heterologous
system, adaptor components from bovine brain CCVs
are reversibly retained on affinity columns prepared from
the C-terminal cystosolic domain of the Arabidopsis
80 kDa homologue ( Z38123) protein. In parallel studies,
adaptor preparations from CCVs prepared from wheat
germ and developing pea cotyledon have also been shown
to bind to the same C-terminal sequence. The binding is
eliminated by preincubating the adaptor preparation with
a truncated terminal sequence containing the tyrosinebased motif (Butler and Beevers, 1997).
The adaptor preparations from developing peas interact
with clathrin-containing components in assembly of coat
structures (Beevers, 1996). Thus, the adaptor fraction
from plants, as those from mammalian systems, by interacting with receptor cytoplasmic tails and clathrin components, provide a rudimentary mechanism for the
selective retrieval of receptors and ligated cargo into
clathrin-coated vesicles. The assembly of coated vesicles
will be much more complex; the basis of selectivity and
clustering of receptors, exclusion of the resident proteins
in the trans-Golgi network, and the nature of lipids
retrieved from the membrane are unresolved. Other components involved in the secretory pathway have been
identified by elegant genetic studies in yeast ( Klionsky
et al., 1990; Stack et al., 1995; Raymond et al., 1992). As
indicated later, complementation studies have identified
plant homologues, but their function in plants is just now
being elucidated.
ATPase with homology to hsp70 ( Kirsch and Beevers,
1993). Such uncoating releases clathrin cage components
(heavy and light chains) for another round of vesicle
assembly. The mechanism of dissociation of adaptors
from receptors, which could release the adaptors from
association with the membranous vesicle and permit their
recycling, is not known. The membranous vesicles, containing receptor and packaged cargo, apparently fuse
with each other or with the prevacuolar compartment
prior to deposition of cargo into the lytic or storage
vacuole.
In mammalian systems, separation of ligand and receptor occurs in a prelysosomal endosomal compartment
and is apparently mediated by a lowering of the pH.
Acidification of the endosomal compartment is apparently
mediated by a vacuolar-type proton pumping ATPase
(Mellman, 1992). The fact that in vitro the receptor was
separated from the NPIR motif ( Kirsch et al., 1994) by
acidification suggests that a similar mechanism operates
in the plant secretory system. Significantly, peptides of
the vacuolar ATPase are present in CCVs, but are inactive
in proton pumping (Drucker et al., 1993; Lin et al.,
1997). Protein pumping by a vacuolar-type ATPase is
demonstrable in the LDM fraction (Lin, 1992; Lin et al.,
1997) and other membranous components (Oberbeck
et al., 1994; Herman et al., 1994). The observation that
bafilomycin A and concanamycin A, inhibitors of vacuolar-type H+-ATPase, disrupt sorting of vacuolar protein
precursors in tobacco cells supports the contention that
vesicle acidification is a necessary component of sorting
in the plant secretory pathway (Matsuoka et al., 1997).
It is suggested that this acidification occurs in a postclathrin membranous fraction, perhaps the provacuole.
From trans-Golgi to the vacuole
Vesicle fusion: SNARE family
Although CCVs provide a mechanism for the selection
of soluble proteins from the secretory system, it does not
appear that they transport their cargo directly to the
vacuole. Cell fractionation studies have identified the
80 kDa protein and the Arabidopsis homologue in a
membrane fraction less dense than CCVs, but not with
the tonoplast ( Kirsch et al., 1994; Ahmed et al., 1997).
Ultrastructural studies demonstrate the occurrence of the
80 kDa protein in a prevacuolar compartment distinct
from CCVs, the trans-Golgi, and the vacuole (Paris et al.,
1997). This compartment may be the functional equivalent in plants of the endosome identified in mammalian
cells and observed in ultrastructural studies of endocytosis
in plants ( Villanueva et al., 1993; Robinson and Hillmer,
1990). Transfer of the 80 kDa protein from CCVs to the
prevacuolar compartment will involve uncoating and
fusion of vesicles into the larger provacuoles. Enzymatic
activity capable of uncoating CCVs has been identified in
extracts from plants. The reaction is mediated by an
As discussed earlier, vesicle budding is mediated by coat
proteins that assemble on nascent buds under the direction
of small GTPases (Schekman and Orci, 1996). After the
vesicle buds off, the coat is removed, and the vesicle
docks with the acceptor compartment in a process
described by the SNARE hypothesis (SNAP receptor
hypothesis) (Söllner et al., 1993). This docking is mediated
by distinct protein families (Pfeffer, 1996). On the vesicle
surface are proteins called v-SNAREs (alternatively,
termed synaptobrevins or VAMPS) that specifically interact with cognate t-SNAREs (or syntaxins) displayed at
the target compartment. The t-SNAREs are maintained
in an inactive state (Pevsner et al., 1994; Lupashin and
Waters, 1997) by association with members of the Sec1like protein family that includes Sly1p, Vps33, and Vps45
(Halachmi and Lev, 1996). Activation of t-SNAREs is
mediated by the displacement of the Sec1-like protein by
a member of the Rab-GTPase family (Lupashin and
Waters, 1997). The characteristic localization of the
The trans-Golgi to vacuole 1275
SNAREs, Rab, and Sec1-like families to distinct
intracellular compartments is thought to reflect the specificity for vesicular trafficking. Following the assembly of
a v- and t-SNARE docking complex, vesicular fusion is
initiated by the interaction of two soluble components,
termed NSF (N-ethylmaleimide-sensitive factor) (Block
et al., 1988) and SNAP (soluble NSF-associated protein)
(Clary et al., 1990). NSF is an ATPase and hydrolysis of
ATP by NSF causes disassembly of the complex, leading
to membrane fusion (Söllner et al., 1993).
Different target membranes and vesicle populations
throughout the cell contain different isoforms of the tSNARE proteins. Each v-SNARE can interact with only
one or a subset of the t-SNAREs. The specificity of fusion
between a vesicle and target membrane could, therefore,
be controlled (at least in part) by the isoforms of the vand t-SNAREs present in the membranes (Söllner et al.,
1993; Rothman, 1994). Many elements of the trafficking
machinery have been found to be conserved among
eukaryotes. For example, v-SNARE and t-SNARE-type
proteins have been isolated from many organisms including yeasts, mammals, insects, and plants (Aalto et al.,
1993; Protopopov et al., 1993; Bassham et al., 1995).
In yeast, the components of the trafficking machinery
required for transport of the vacuolar hydrolase
carboxypeptidase Y (CPY ) from Golgi to the vacuole
have been identified. This machinery includes many components previously identified for other vesicle trafficking
steps. Vesicles that carry CPY are thought to contain the
v-SNARE Vti1p ( Fischer von Mollard et al., 1997) which
directs delivery to the prevacuole/endosome through
interaction with the endosomal t-SNARE Pep12p and
several soluble factors including NSF and the Sec1phomologue, Vps45 (Burd et al., 1997; Fischer von Mollard
et al., 1997). CPY is likely released from its receptor,
Vps10p at this point, perhaps due to the lower pH of the
endosomal compartment (Marcusson et al., 1994). CPY
then continues on to the vacuole through a process that
requires the vacuolar t-SNARE Vam3p (Darsow et al.,
1997), while Vps10p and Vtip are recycled back to the
Golgi apparatus (Cereghino et al., 1995; Cooper and
Stevens, 1996; Fischer von Mollard et al., 1997).
Although considerable research has been done on
defining the trafficking machinery in yeast and mammalian cells, very little is known about the mechanics of
trafficking in plants. A functional homologue of a yeast
syntaxin involved in vacuolar transport (PEP12) has been
isolated from Arabidopsis thaliana (AtPEP12) as a cDNA
that complements a yeast pep12 deletion mutant
(Bassham et al., 1995). The cDNA isolated in this screen
is predicted to encode a protein homologous to PEP12
and mammalian syntaxins. It is likely that this protein
represents an essential part of the plant vacuolar targeting
machinery.
Like Pep12p and other syntaxins, AtPEP12p contains
a C-terminal coiled-coil domain and a highly conserved
C-terminal membrane anchor domain (59% homologous
to Pep12p). Northern analysis has shown that a single
1.3 kb mRNA is expressed in plants; the highest level is
found in roots, stems and flowers, with lower levels found
in leaves (Bassham et al., 1995). Antibodies have been
produced in rabbits to the bacterially overexpressed
hydrophilic N-terminus of AtPEP12p that immunoprecipitate in vitro translated AtPEP12p as a single band of
~35 kDa. Use of this antiserum in Western analysis of
several plant tissues shows, similarly to what was found
for AtPEP12 mRNA, that the highest amount of
AtPEP12p is found in roots, with lower levels in leaves
and stems. Interestingly, either one or a range of two to
three bands was recognized by the AtPEP12 antibodies,
depending on the tissues analysed. Three bands are
observed in roots and cell suspension cultures, whereas
in leaves and siliques the antibodies recognize a single
major band of 36 kDa. This suggests that AtPEP12p is
either post-translationally modified or that a second group
of proteins different from AtPEP12p, is cross-reactive
with AtPEP12 antibodies; both possibilities may occur in
different tissues. Biochemical fractionation indicates that
the AtPEP12p is found exclusively associated with cellular membranes, as expected for an integral membrane
protein. Various gradient centrifugations show that
AtPEP12p does not cofractionate with membrane
markers available for the Arabidopsis ER (AtSEC12p;
Bar-Peled and Raikhel, 1997), Golgi ( latent-IDPase and
fucosyltransferase), tonoplast (c-TIP), or plasma membrane (RD48; Conceição et al., 1997). It is proposed to
call this AtPEP12-containing fraction a prevacuolar compartment (Anton Sanderfoot and Natasha Raikhel,
unpublished data). Precise designation of these membranes as arising from prevacuolar compartments can be
made if cargo destined for the vacuole (proteins with
vacuolar-targeting signals) is found associated with these
AtPEP12p-vesicles. A question that should be resolved is
whether the vacuolar proteins, using three different targeting signals, are delivered to the vacuole via the same
compartment carrying AtPep12p, or whether some other
unidentified syntaxin proteins associated with other vesicles are responsible for the delivery of a subset of
vacuolar proteins. Also, identification of other endomembrane markers, localization of which overlaps with this
AtPEP12p containing membrane, will allow us to evaluate
the exact nature of this organelle better and to understand
the complexity and specificity of the vacuolar pathway
in plants.
Another syntaxin, AtVAM3p, Arabidopsis homologue
of the yeast vacuolar-t-SNARE Vam3p, has been identified by functional complementation (Sato et al., 1977).
The AtVAM3p is 60% identical to AtPEP12p and both
genes share similar patterns of expression. Electron microscopic analysis of apical shoot meristem reveals that
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Beevers and Raikhel
AtVAM3p is localized to small patches on the tonoplast
where two vacuoles are connected, suggesting that
AtVAM3p functions in membrane–membrane interactions (Sato et al., 1997). The localization of AtPEP12p
and AtVAMp has been performed in different tissues;
AtPEP12p has been localized in roots and AtVAM3p in
apical shoot meristem. Considering the homology
between AtPEP12p and AtVAM3p (60% identity) but
their different apparent subcellular localization, it will be
crucial to investigate the distribution of both proteins in
the same cell types.
Vps45p is a Sec1p-like protein that is required for
transport between the TGN and prevacuole/endosome in
yeast (Piper et al., 1994; Cowles et al., 1994). Recently,
a cDNA encoding an Arabidopsis VPS45 homologue,
AtVPS45, has been isolated (Bassham and Raikhel,
1998). AtVPS45 complements the yeast vps45 mutant,
indicating that the two proteins are functionally related.
AtVPS45p is a peripheral membrane protein that associates with low-density membranes. Sucrose gradient fractionation demonstrates that AtVPS45p co-fractionates
with a potential vacuolar targeting receptor, AtELP,
implying that they may reside on the same membrane
population. In addition, AtVPS45p partially cofractionates with AtPEP12p, prevacuolar syntaxin homologue. These observations indicate that AtVPS45p is likely
to function in the transport of proteins to the vacuole in
plants (Bassham and Raikhel, 1998).
Other genes presumably involved in vacuolar protein
sorting have been isolated by homology to yeast genes.
A PI 3-kinase gene has been isolated from Arabidopsis
showing some sequence homology to the yeast VPS34
gene and to mammalian PI 3-kinase ( Welters et al., 1994).
Although overexpression of the AtVP3S4 gene in transgenic plants indicates that the gene encodes a functional
PI 3-kinase, the AtVPS34 does not complement a yeast
vps34 mutant. The function of AtVPS34 in planta is not
yet known.
An Arabidopsis gene showing homology to the yeast
VPS1 gene, which encodes a GTP-binding protein
required for localization of soluble hydrolases to the
vacuole, has also been identified (Dombrowski and
Raikhel, 1995), although it has not been demonstrated
that the plant homologue functions in vacuolar targeting.
Several small GTP-binding proteins have been found to
be associated with vesicles carrying vacuolar proteins in
pumpkin cotyledon cells, and their function in protein
transport has been proposed (Shimada et al., 1994).
Another small GTP-binding protein, Rab6, presumably
functions in the regulation of vesicle transport from the
TGN in mammals. The Arabidopsis homologue of that
gene has also been cloned (Bednarek et al., 1994).
AtRAB6 has approximately 79% sequence identity to the
mammalian (Rab6) and yeast (Ryh1 and Ypt6) protein
counterparts. Although the AtRAB6 functionally comple-
ments the YPT6 mutant from yeast, its function in vivo
is not yet known.
Conclusions
The transport of soluble proteins through the plant
secretory pathway to the vacuole has been studied in
some detail at the level of the targeting signals within the
protein, and as this review illustrates, progress has been
made in identifying receptors and potential transport
vesicles. However, many major questions remain about
the mechanisms by which the proteins are transported
(Bar-Peled et al., 1996). Several components of the vesicle
transport machinery have been isolated, but their precise
function in planta remains to be addressed. In addition,
the use of genetic and biochemical approaches will be
essential for isolating further components of the vesicle
targeting machinery in the near future. Only when the
majority of the markers to enable differentiation between
the various components of the secretory machinery have
been identified will it be possible to address the differences
between the various cell types and tissues mechanistically.
Some proteins are likely to follow a similar path to the
vacuole in plant cells as in yeast; however, plants are
multicellular organisms composed of many different tissues and cell types. In some tissues individual cells appear
to have discrete plant vacuoles which serve either a
storage or a lytic function; in other situations, some
vacuoles are multifunctional organelles accumulating
storage proteins, hydrolases, and varieties of defence
molecules (Matile, 1975). Undoubtedly, vesicular
trafficking to the multiplicity of vacuoles with differing
functions in plants will be more complex than in yeast.
For most of the homologues in yeast trafficking machinery
that are governed by only one gene, two or more proteins
have been found in plants. Indeed, the existence of several
AtPEP12p-like proteins in Arabidopsis (Hayian Zheng
and Natasha Raikhel, unpublished results) is consistent
with the idea that a single protein in yeast may have
several isoforms in plants. Multiple cDNAs for potential
isoforms of vacuolar receptor proteins and components
of clathrin adaptors have been identified. It may be that
the multiplicity of genes and isoform proteins merely
represent redundancy. However, it is tempting to speculate that the diversity of isoforms for specific proteins
may provide the flexibility for selectivity for delivery of
proteins from the secretory system to a variety of
destinations.
Acknowledgements
The research was supported by grants from the National
Science Foundation (grant No. MCB-9507030 and the US
Department of Energy (grant No. DE-FG02-91ER20021) to
The trans-Golgi to vacuole 1277
NVR and from the National Science Foundation (grant No.
MCB-9304758) to LB.
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